Cetuximab (CTX) is a potent chimeric mouse/human monoclonal antibody (mAb) approved worldwide for treatment of metastatic colorectal cancer. Among the various biological and physical analyses per- formed for full ...Cetuximab (CTX) is a potent chimeric mouse/human monoclonal antibody (mAb) approved worldwide for treatment of metastatic colorectal cancer. Among the various biological and physical analyses per- formed for full study on this biopharmaceutic, the determination of the concentration preparations throughout manufacturing and subsequent handling in hospital is particularly relevant. In the present work, the study and validation of a method for quantifying intact CTX by reverse-phase high-perfor- mance liquid chromatography with diode array detection ((RP)HPLC/DAD) is presented. With that end, we checked the performance of a chromatographic method for quantifying CTX and conducted a study to validate the method as stability-indicating in accordance with the International Conference on Harmo- nization guidelines (ICH) for biotechnological drugs; therefore, we evaluated linearity, accuracy, preci- sion, detection and quantification limits, robustness and system suitability. The specificity of the method and the robustness of the mAb formulation against external stress factors were estimated by compre- hensive chromatographic analysis by subjecting CTX to several informative stress conditions. As de- monstrated, the method is rapid, accurate, and reproducible for CTX quantification. It was also suc- cessfully used to quantify CTX in a long-term stability study performed under hospital conditions.展开更多
The research was carried out for establishing a new reverse phase-HPLC stability indicating method for the quantification of Rucaparib. The experiment was determined on Waters HPLC instrument using 996 photo-diode arr...The research was carried out for establishing a new reverse phase-HPLC stability indicating method for the quantification of Rucaparib. The experiment was determined on Waters HPLC instrument using 996 photo-diode array detector. The separation was done by using symmetry C-18 ODS (25 cm × 0.46 cm internal diameter) 5 μm analytical column containing mobile phase of Phosphate buffer (0.02 M) and methanol [65:35% v/v] adjusted pH to 4.8 by adding dilute ortho phosphoric acid. The method was run at 1 ml·min<sup>-1</sup> at 286 nm detection. The drug was eluted at 5.484 min. After developing the method, it was assured for the intended use by validation which was done according to ICH Q2B guidelines. The analytical parameters checked were linearity, accuracy, repeatability, intermediate precision, limit of detection, limit of quantitation, ruggedness and robustness. It was observed that the response of the detector was linear in the range of 6 - 14 μg/ml with correlation coefficient of 0.999. The results of all the parameters were found to be within the acceptance criteria. The stability indicating assay method was established by using the samples generated by forced degradation process. The forced degradation was carried out by subjecting the drug to acid, alkali, thermal, oxidative and photolytic degradation and the results showed that the degradation products were successfully separated from the drug. Hence, this can be applied perfectly later for the analysis of quality of the rucaparib drug.展开更多
[Objectives]This paper aims to establish a method to simultaneously determine the content ofβ-asarone in Rhizoma Acori Tatarinowii dried by three different methods.[Methods]Reversed-phase high-performance liquid chro...[Objectives]This paper aims to establish a method to simultaneously determine the content ofβ-asarone in Rhizoma Acori Tatarinowii dried by three different methods.[Methods]Reversed-phase high-performance liquid chromatography was used,and the chromatographic conditions were as follows:column,Thermo SCIENTIFIC Hypersil GOLD Dim.(mm);mobile phase,methanol-0.1%phosphoric acid(63∶37);column temperature,30℃;flow rate,1mL/min;detection wavelength,257 nm;sample size,10μL.[Results]The linear range of the injection volume ofβ-asarone was 49.28-246.40μg/mL(R=0.9993);the limit of quantification was 0.85 ng and the detection limit was 0.34 ng;the RSD values of precision,stability and reproducibility tests were all less than 3%;and the sample recovery rate was 98.53%-98.97%(RSD<3.00).The results show that the content ofβ-asarone was highest in shade-dried Rhizoma Acori Tatarinowii.The order ofβ-asarone content was as follows:Rhizoma Acori Tatarinowii dried in shade>Rhizoma Acori Tatarinowii dried at 55℃>Rhizoma Acori Tatarinowii dried at 60℃.[Conclusions]This method is sensitive,reliable,and reproducible.It can be used to simultaneously determine the content ofβ-asarone in Rhizoma Acori Tatarinowii dried by three different methods.展开更多
The research was carried out to establish a new reverse phase-HPLC stability indicating method for quantifying Bimatoprost & Timolol in ophthalmic solution. The experiment of Bimatoprost & Timolol in ophthalmi...The research was carried out to establish a new reverse phase-HPLC stability indicating method for quantifying Bimatoprost & Timolol in ophthalmic solution. The experiment of Bimatoprost & Timolol in ophthalmic solution method development was determined on Waters HPLC instrument using a UV Detector. The separation was done by using L11, Zorbex SB phenyl (4.6 mm × 250 mm internal diameter) 5 μm analytical column, containing mobile phase of Phosphate buffer (0.02 M), methanol, and acetonitrile [50:30:20 % v/v]. The method was run at 1 ml·min<sup>-1</sup> at 210 nm for Bimatoprost and 295 nm for Timolol for detection. The drug was eluted at 10.81 min for Bimatoprost and 3.77 min for Timolol. After developing the method, it was assured for the intended use by validation, which was done according to ICH Q2B guidelines. The analytical parameters checked were Specificity/Selectivity, linearity, Range, accuracy, ruggedness, and robustness. It was observed that the response of the detector was linear in the range of 6 - 18 μg/ml with a correlation coefficient of 0.999. The results of all the parameters were found to be within the acceptance criteria. The stability-indicating assay method was established by using the samples generated by the forced degradation process. The forced degradation was carried out by subjecting the drug to acid, alkali, thermal, oxidative, and photolytic degradation, and the results showed that the degradation products were successfully separated from the drug. Hence, this can be applied perfectly later for the quantitative analysis of Bimatoprost 0.3% + Timolol 0.5% Ophthalmic Solution drugs for pharmaceutical use. Currently, there is no official method for Bimatoprost & Timolol combination products in USP or BP. Available research work related to single Bimatoprost or Timolol products was not suitable for testing Bimatoprost and Timolol combination drugs. Additionally, there is no stability-indicating method to test Bimatoprost & Timolol combination products which insist us to do research and develop a new reverse phase-HPLC indicating method which will be faster and more accurate.展开更多
One antibacterial activity fraction from an immunized dipteran insect, Bactrocera dorsalis, was isolated and purified by prepurification, ion-exchange chromatography, gel filtration chromatography and reverse-phase hi...One antibacterial activity fraction from an immunized dipteran insect, Bactrocera dorsalis, was isolated and purified by prepurification, ion-exchange chromatography, gel filtration chromatography and reverse-phase high performance liquid chromatography (HPLC). The final purified fraction was checked on the Smart system HPLC and was judged as a pure fraction. The results of physical and biological analysis revealed that this fraction is heat stable and showed strong activities against Gram-positive bacterial growth. It possesses antibicrobial peptide properties and is worth further investigation.展开更多
A method was established to determine the activity of hexokinase by reverse-phase high-performance liquid chromatography (RP-HPLC). The activity of hexokinase was determined by detecting the amount of ADP in the rea...A method was established to determine the activity of hexokinase by reverse-phase high-performance liquid chromatography (RP-HPLC). The activity of hexokinase was determined by detecting the amount of ADP in the reaction that converts glucose into glucose-6-phosphate. The separation degree of ATP and ADP was as good as 3.46 (separation degree^l.5). In the range of 0.01-0.40 mmol/L, there was a good linear relationship between concentration of ADP and its peak areas (the correlation coefficient was 0.999). The relative standard deviation(RSD) of intraday was 1.43%-1.46%, that of interday was 2.12%-2.15%. At 30℃ (optimal temperature) and pH7.0 (optimal pH), the recovery rate of hexokinase was 95.3%-97.6%. The results showed that the method had good precision and high recovery rate, and the enzyme activity was determined through only one-step reaction. To some extent, this method can reduce the cost.展开更多
基金funded by Project FIS:PI10/00201 (Instituto Carlos III, Ministerio de Economía y Competitividad, Spain)partially supported by European Regional Development Funds (ERDF)
文摘Cetuximab (CTX) is a potent chimeric mouse/human monoclonal antibody (mAb) approved worldwide for treatment of metastatic colorectal cancer. Among the various biological and physical analyses per- formed for full study on this biopharmaceutic, the determination of the concentration preparations throughout manufacturing and subsequent handling in hospital is particularly relevant. In the present work, the study and validation of a method for quantifying intact CTX by reverse-phase high-perfor- mance liquid chromatography with diode array detection ((RP)HPLC/DAD) is presented. With that end, we checked the performance of a chromatographic method for quantifying CTX and conducted a study to validate the method as stability-indicating in accordance with the International Conference on Harmo- nization guidelines (ICH) for biotechnological drugs; therefore, we evaluated linearity, accuracy, preci- sion, detection and quantification limits, robustness and system suitability. The specificity of the method and the robustness of the mAb formulation against external stress factors were estimated by compre- hensive chromatographic analysis by subjecting CTX to several informative stress conditions. As de- monstrated, the method is rapid, accurate, and reproducible for CTX quantification. It was also suc- cessfully used to quantify CTX in a long-term stability study performed under hospital conditions.
文摘The research was carried out for establishing a new reverse phase-HPLC stability indicating method for the quantification of Rucaparib. The experiment was determined on Waters HPLC instrument using 996 photo-diode array detector. The separation was done by using symmetry C-18 ODS (25 cm × 0.46 cm internal diameter) 5 μm analytical column containing mobile phase of Phosphate buffer (0.02 M) and methanol [65:35% v/v] adjusted pH to 4.8 by adding dilute ortho phosphoric acid. The method was run at 1 ml·min<sup>-1</sup> at 286 nm detection. The drug was eluted at 5.484 min. After developing the method, it was assured for the intended use by validation which was done according to ICH Q2B guidelines. The analytical parameters checked were linearity, accuracy, repeatability, intermediate precision, limit of detection, limit of quantitation, ruggedness and robustness. It was observed that the response of the detector was linear in the range of 6 - 14 μg/ml with correlation coefficient of 0.999. The results of all the parameters were found to be within the acceptance criteria. The stability indicating assay method was established by using the samples generated by forced degradation process. The forced degradation was carried out by subjecting the drug to acid, alkali, thermal, oxidative and photolytic degradation and the results showed that the degradation products were successfully separated from the drug. Hence, this can be applied perfectly later for the analysis of quality of the rucaparib drug.
基金Key Research and Development Project of Department of Science and Technology of Guangxi Zhuang Autonomous Region(AB19110003)Project for Improving Basic Scientific Research Ability of Young and Middle-aged Teachers in Colleges and Universities of Guangxi(2019KY0341,2019ky0344)+2 种基金Open Project of Guangxi Zhuang Yao Medicine Center of Engineering and Technology(KJT1900105)Youth Foundation of Guangxi University of Chinese Medicine(2019QN036,2019QN030)Traditional Chinese Medicine Scientific Research Laboratory(Grade III)of National Administration of Traditional Chinese Medicine:Laboratory of Chinese(Zhuang)Medicine Chemical and Quality Analysis(Guo Zhong Yi Yao Fa 2009[21]).
文摘[Objectives]This paper aims to establish a method to simultaneously determine the content ofβ-asarone in Rhizoma Acori Tatarinowii dried by three different methods.[Methods]Reversed-phase high-performance liquid chromatography was used,and the chromatographic conditions were as follows:column,Thermo SCIENTIFIC Hypersil GOLD Dim.(mm);mobile phase,methanol-0.1%phosphoric acid(63∶37);column temperature,30℃;flow rate,1mL/min;detection wavelength,257 nm;sample size,10μL.[Results]The linear range of the injection volume ofβ-asarone was 49.28-246.40μg/mL(R=0.9993);the limit of quantification was 0.85 ng and the detection limit was 0.34 ng;the RSD values of precision,stability and reproducibility tests were all less than 3%;and the sample recovery rate was 98.53%-98.97%(RSD<3.00).The results show that the content ofβ-asarone was highest in shade-dried Rhizoma Acori Tatarinowii.The order ofβ-asarone content was as follows:Rhizoma Acori Tatarinowii dried in shade>Rhizoma Acori Tatarinowii dried at 55℃>Rhizoma Acori Tatarinowii dried at 60℃.[Conclusions]This method is sensitive,reliable,and reproducible.It can be used to simultaneously determine the content ofβ-asarone in Rhizoma Acori Tatarinowii dried by three different methods.
文摘The research was carried out to establish a new reverse phase-HPLC stability indicating method for quantifying Bimatoprost & Timolol in ophthalmic solution. The experiment of Bimatoprost & Timolol in ophthalmic solution method development was determined on Waters HPLC instrument using a UV Detector. The separation was done by using L11, Zorbex SB phenyl (4.6 mm × 250 mm internal diameter) 5 μm analytical column, containing mobile phase of Phosphate buffer (0.02 M), methanol, and acetonitrile [50:30:20 % v/v]. The method was run at 1 ml·min<sup>-1</sup> at 210 nm for Bimatoprost and 295 nm for Timolol for detection. The drug was eluted at 10.81 min for Bimatoprost and 3.77 min for Timolol. After developing the method, it was assured for the intended use by validation, which was done according to ICH Q2B guidelines. The analytical parameters checked were Specificity/Selectivity, linearity, Range, accuracy, ruggedness, and robustness. It was observed that the response of the detector was linear in the range of 6 - 18 μg/ml with a correlation coefficient of 0.999. The results of all the parameters were found to be within the acceptance criteria. The stability-indicating assay method was established by using the samples generated by the forced degradation process. The forced degradation was carried out by subjecting the drug to acid, alkali, thermal, oxidative, and photolytic degradation, and the results showed that the degradation products were successfully separated from the drug. Hence, this can be applied perfectly later for the quantitative analysis of Bimatoprost 0.3% + Timolol 0.5% Ophthalmic Solution drugs for pharmaceutical use. Currently, there is no official method for Bimatoprost & Timolol combination products in USP or BP. Available research work related to single Bimatoprost or Timolol products was not suitable for testing Bimatoprost and Timolol combination drugs. Additionally, there is no stability-indicating method to test Bimatoprost & Timolol combination products which insist us to do research and develop a new reverse phase-HPLC indicating method which will be faster and more accurate.
基金Acknowledgments We thank Prof. X.-F. Pang, late Academician of Chinese Academy of Science, for his support in getting this project started. We also thank Prof. Yamakawa (National of Institute of Sericultural and Entomological Science, Japan) for analyzing the purity of the final purified fraction. We thank Dr H. Ma for his suggestions on this project and reading our manuscript. This work was supported partly by the Natural Science Foundation of Guangdong Province (032256), the National Basic Research Program of China under grant 2005CB121000 and the National Natural Science Foundation (30370716).
文摘One antibacterial activity fraction from an immunized dipteran insect, Bactrocera dorsalis, was isolated and purified by prepurification, ion-exchange chromatography, gel filtration chromatography and reverse-phase high performance liquid chromatography (HPLC). The final purified fraction was checked on the Smart system HPLC and was judged as a pure fraction. The results of physical and biological analysis revealed that this fraction is heat stable and showed strong activities against Gram-positive bacterial growth. It possesses antibicrobial peptide properties and is worth further investigation.
基金Supported by the Key Project of Science and Technology Department of Fujian Province (2008Y0052)
文摘A method was established to determine the activity of hexokinase by reverse-phase high-performance liquid chromatography (RP-HPLC). The activity of hexokinase was determined by detecting the amount of ADP in the reaction that converts glucose into glucose-6-phosphate. The separation degree of ATP and ADP was as good as 3.46 (separation degree^l.5). In the range of 0.01-0.40 mmol/L, there was a good linear relationship between concentration of ADP and its peak areas (the correlation coefficient was 0.999). The relative standard deviation(RSD) of intraday was 1.43%-1.46%, that of interday was 2.12%-2.15%. At 30℃ (optimal temperature) and pH7.0 (optimal pH), the recovery rate of hexokinase was 95.3%-97.6%. The results showed that the method had good precision and high recovery rate, and the enzyme activity was determined through only one-step reaction. To some extent, this method can reduce the cost.